Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System
Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would...
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doaj-9262a0e728444d9b8fe44521cb2df8bb2020-11-24T23:24:23ZengHindawi LimitedBioMed Research International2314-61332314-61412016-01-01201610.1155/2016/34236853423685Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression SystemMohammed N. Baeshen0Thamer A. F. Bouback1Mubarak A. Alzubaidi2Roop S. Bora3Mohammed A. T. Alotaibi4Omar T. O. Alabbas5Sultan M. Alshahrani6Ahmed A. M. Aljohani7Rayan A. A. Munshi8Ahmed Al-Hejin9Mohamed M. M. Ahmed10Elrashdy M. Redwan11Hassan A. I. Ramadan12Kulvinder S. Saini13Nabih A. Baeshen14Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaDepartment of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaDepartment of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaDepartment of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaDepartment of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaDepartment of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaDepartment of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaDepartment of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaDepartment of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaDepartment of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaDepartment of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaDepartment of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaDepartment of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaDepartment of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaDepartment of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi ArabiaIncrease in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system.http://dx.doi.org/10.1155/2016/3423685 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Mohammed N. Baeshen Thamer A. F. Bouback Mubarak A. Alzubaidi Roop S. Bora Mohammed A. T. Alotaibi Omar T. O. Alabbas Sultan M. Alshahrani Ahmed A. M. Aljohani Rayan A. A. Munshi Ahmed Al-Hejin Mohamed M. M. Ahmed Elrashdy M. Redwan Hassan A. I. Ramadan Kulvinder S. Saini Nabih A. Baeshen |
spellingShingle |
Mohammed N. Baeshen Thamer A. F. Bouback Mubarak A. Alzubaidi Roop S. Bora Mohammed A. T. Alotaibi Omar T. O. Alabbas Sultan M. Alshahrani Ahmed A. M. Aljohani Rayan A. A. Munshi Ahmed Al-Hejin Mohamed M. M. Ahmed Elrashdy M. Redwan Hassan A. I. Ramadan Kulvinder S. Saini Nabih A. Baeshen Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System BioMed Research International |
author_facet |
Mohammed N. Baeshen Thamer A. F. Bouback Mubarak A. Alzubaidi Roop S. Bora Mohammed A. T. Alotaibi Omar T. O. Alabbas Sultan M. Alshahrani Ahmed A. M. Aljohani Rayan A. A. Munshi Ahmed Al-Hejin Mohamed M. M. Ahmed Elrashdy M. Redwan Hassan A. I. Ramadan Kulvinder S. Saini Nabih A. Baeshen |
author_sort |
Mohammed N. Baeshen |
title |
Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System |
title_short |
Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System |
title_full |
Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System |
title_fullStr |
Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System |
title_full_unstemmed |
Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System |
title_sort |
expression and purification of c-peptide containing insulin using pichia pastoris expression system |
publisher |
Hindawi Limited |
series |
BioMed Research International |
issn |
2314-6133 2314-6141 |
publishDate |
2016-01-01 |
description |
Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system. |
url |
http://dx.doi.org/10.1155/2016/3423685 |
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